This invention relates to the fields of vaccination and diagnostics in connection with diseases which are caused by pathogens and involves the use of both the classic methods to arrive at a live attenuated vaccine or an inactivated vaccine and the modern methods based on DNA recombinant technology.
More specifically, the invention relates to live attenuated vaccines an inactivated vaccines for protecting animals, especially bovines, against bovine herpesvirus type 1 (BHV-1), these vaccines being so designed that they are not only safe and effective, but also create the possibility of distinguishing infected from non-infected animals in a vaccinated population.
Diagnostic kits which can be used for such a test for distinguishing infected from non-infected animals in a vaccinated population are also an aspect of the present invention.
BHV-1, including the infectious bovine rhinotracheitis virus (IBRV) and the infectious pustular vulvovaginitis virus (IPVV), plays an important role in the development of respiratory diseases and fertility disorders in bovines. After an acute infection, BHV-1 often remains present in the host in a latent form. Latent virus can be reactivated under the influence of inter alia stressxe2x80x94which may or may not be accompanied by clinical phenomenaxe2x80x94and subsequently excreted. As a consequence, infected cattle must be regarded as lifelong potential spreaders of BHV-1. BHV-1 occurs endemically in an estimated 75% of Dutch cattle farms. Especially older cattle are serologically positive.
There are a number of inactivated (xe2x80x9cdeadxe2x80x9d) vaccines and a number of attenuated (xe2x80x9clivexe2x80x9d) vaccines available for inoculation against BHV-1 infections. Inactivated vaccines are prepared by killing the BHV-1 virus, for instance by heat treatment, irradiation or treatment with ethanol or formalin. However, these often give insufficient protection. Attenuated vaccines are prepared by a large number of passages on homologous (bovine) or on heterologous cells such as porcine or canine cells, and sometimes viruses are also treated physically or chemically then. In this way, unknown mutations/deletions develop in the virus genome, which often reduce the disease-producing properties of the virus. Attenuated live vaccines give better protection than inactivated vaccines, inter alia because they present more viral antigens to the immune system of the host. Another important advantage of live vaccines is that they can be administered intranasally, i.e., at the site where the first multiplication of the wild type virus occurs after infection. Yet, live vaccines leave room for improvement. Some live vaccines still seem to possess their abortogenic ability, which becomes manifest in particular after intramuscular administration. Moreover, probably all live vaccines remain latently present in the vaccinated cow. Also, there is a chance that if the vaccine differs only little from the wild-type virus, reversion to virulence will occur. But one of the major problems is that the BHV-1 vaccines cannot prevent infection by wild-type viruses. The result is that vaccinated cattle can also spread wild-type BHV-1.
For a proper BHV-1 control program, it is necessary to have disposal of an efficacious and safe vaccine that can be distinguished from wild-type virus, since the application of an efficacious vaccine can reduce the circulation of BHV-1 considerably and a test which can distinguish between a vaccine and a wild-type virus makes it possible to detect (and then remove) infected cattle in a vaccinated population.
Meanwhile, BHV-1 vaccines have been developed which seem to be safer than conventional vaccines and are distinguishable from wild-type virus. A thymidine kinase deletion mutant has been isolated which is abortogenic to a lesser degree, becomes latent less frequently and cannot be reactivated. Further, using recombinant DNA techniques, a BHV-1 vaccine has been constructed which has a deletion in the gene for glycoprotein gIII, which makes this vaccine distinguishable from wild-type BHV-1 by means of serological techniques. However, there are still some objections to these vaccines. On the one hand, the thymidine kinase gene is involved in the viral replication and less replication can lead to less protection. On the other hand, the glycoprotein gIII is important for generating protective antibodies, which makes a gIII deletion vaccine less effective. A practical problem is that intranasal administration, which generally gives the best protection, of recombinant vaccines is not allowed in some countries. Accordingly, there is a need for a vaccine which is safe as well as effective and yet can be distinguished from wild-type BHV-1, it being further desirable that at least one of such vaccines is based on a virus attenuated via a conventional route rather than a virus constructed by recombinant DNA techniques.
Now, via passages in cell cultures, a BHV-1 strain has been obtained which lacks the gene for glycoprotein gE. The first results of our research indicate that this gene is quite useful to make a serological distinction with regard to wild-type BHV-1 and that it is involved in the expression of virulence. Therefore, its deletion contributes to safety and may render the use of thymidine kinase deletions superfluous. The glycoprotein gE seems to be less important for induction of protection than the glycoprotein gIII. A conventionally attenuated BHV-1 strain which can be serologically distinguished from wild-type virus is unique. The location and DNA sequence of the gE gene described herein for the first time were not previously known, nor were oligonucleotides, polypeptides and oligopeptides that can be derived therefrom. A test for making a serological distinction on the basis of the gE gene is also unique.
An important advantage of this xe2x80x9cconventionalxe2x80x9d gE deletion mutant (xe2x80x9cconventionalxe2x80x9d refers to the use of a conventional method for isolating an attenuated virus) is that it will be possible to administer it intranasally in countries where this is forbidden as far as recombinant vaccines are concerned. Taking due account of the different views on safety, however, in addition to this conventional gE deletion vaccine, well-defined recombinant versions have been constructed as well. These recombinant vaccines also have a gE deletionxe2x80x94and may or may not have a deletion in the thymidine kinase gene as wellxe2x80x94and can also be used as vectors for the expression of heterologous genes. All these recombinant vaccines can be distinguished from wild-type virus with the same gE-specific test. The use of a standard test for a set of different vaccines can be a great advantage in the combat of BHV-1 as an international effort. Such an approach has not been previously described in the field of BHV-1, vaccines.
Serological analysis of the anti BHV-1 response in cattle showed that an important fraction of the anti-gE antibodies are directed against a complex formed by glycoprotein gE and another BHV-1 glycoprotein: glycoprotein gI. Serological tests that can (also) demonstrate the presence of such complex-specific antibodies may therefore be more sensitive than tests that can only detect anti-gE antibodies. Cattle vaccinated with a single gE deletion mutant may produce anti-gI antibodies that can interfere with the detection of anti-gI/gE antibodies. Consequently, this invention also includes a vaccine with a gI/gE double deletion.
In the first place, this invention provides a deletion mutant of BHV-1 which has a deletion in the glycoprotein gE-gene. The words xe2x80x9ca deletion inxe2x80x9d intended to cover a deletion of the gene as a whole.
A preferred embodiment of the invention is constituted by a deletion mutant of BHV-1 which has a deletion in the glycoprotein gE-gene which has been caused by an attenuation procedure, such as the deletion mutant Difivac-1 to be described hereinafter.
Other preferred embodiments of the invention consist of a deletion mutant of BHV-1 comprising a deletion in the glycoprotein gE-gene which has been constructed by recombinant DNA techniques, such as the deletion mutants 1B7 or 1B8 to be described hereinafter.
Another preferred embodiment of the invention consists of a double deletion mutant of BHV-1 comprising a deletion of the glycoprotein gE-gene and a deletion in the glycoprotein gI-gene, such as the gI/gE double deletion mutant Difivac-IE to be described hereinafter.
Further, with a view to maximum safety, according to the invention a deletion mutant of BHV-1 is preferred which has a deletion in the glycoprotein gE-gene and a deletion in the thymidine kinase gene. The invention also covers a deletion mutant of BHV-1 which has a deletion in the glycoprotein gE-gene, the glycoprotein gI-gene and the thymidine kinase gene.
The invention provides a vaccine composition for vaccination of animals, in particular mammals, more particularly bovines, to protect them against BHV-1, comprising a deletion mutant of BHV-1 as defined hereinabove, and a suitable carrier or adjuvant. Said composition may be a live or an inactivated vaccine composition.
The invention is further embodied in a mutant of BHV-1 which has a deletion in the glycoprotein gE-gene and contains a heterologous gene introduced by recombinant DNA techniques.
Preferably, this concerns a mutant of BHV-1 which contains a heterologous gene introduced by recombinant DNA techniques at the location of the glycoprotein gE-gene, which heterologous gene is under the control of regulatory sequences of the gE-gene and is optionally attached to the part of the gE-gene which codes for a signal peptide. Said heterologous gene may also be under the control of a different promoter of BHV-1, or under the control of a heterologous promoter. When the mutant of BHV-1 has further deletions in addition to the deletion in the glycoprotein gE-gene, such as a deletion in the thymidine kinase gene and/or a deletion in the glycoprotein gI-gene, said heterologous gene may also be inserted at the location of this additional deletion(s). Plural insertions are another option, either together at the location of one deletion, or distributed over locations of several deletions.
The heterologous gene introduced preferably codes for an immunogenic protein or peptide of another pathogen, or for a cytokine which promotes the immune response. Examples of suitable cytokines are interleukin 2, interferon-alpha and interferon-gamma.
The invention also provides a (live or inactivated) vaccine composition for vaccination of animals, in particular mammals, more particularly bovines, to protect them against a (different) pathogen, comprising a mutant of BHV-1 havingxe2x80x94therein a heterologous gene coding for an immunogenic protein or peptide of that other pathogen, and a suitable carrier of adjuvant. Of course, the protection may concern more than one pathogen, i.e. a multivalent vaccine wherein the mutant contains a plurality of heterologous genes.
The invention further relates to a composition comprising a recombinant nucleic acid comprising the glycoprotein gE-gene of BHV-1, a part of this glycoprotein gE-gene or a nucleotide sequence derived from this glycoprotein gE-gene. This composition can contain a cloning or expression vector having therein an insertion of a recombinant nucleic acid which comprises the glycoprotein gE-gene of BHV-1, a part of this glycoprotein gE-gene or a nucleotide sequence derived from this glycoprotein gE-gene.
The invention also comprises a composition comprising glycoprotein gE of BHV-1, a part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex of the glycoproteins gE and gI, and a composition comprising an antibody which is specific for glycoprotein gE of BHV-1, a part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex of the glycoproteins gE and gI. xe2x80x9cAntibodyxe2x80x9d is understood to mean both a polyclonal antibody preparation and a monoclonal antibody preferred for most applications. The terms xe2x80x9ca part of glycoprotein gExe2x80x9d and xe2x80x9ca peptide derived from glycoprotein gExe2x80x9d are understood to mean gE-specific amino acid sequences which generally will have a length of at least about 8 amino acids.
The invention further relates to a diagnostic kit for detecting nucleic acid of BHV-1 in a sample, in particular a biological sample such as blood or blood serum, blood cells, milk, bodily fluids such as tears, lung lavage fluid, nasal fluid, sperm, in particular seminal fluid, saliva, sputum, or tissue, in particular nervous tissue, coming from an animal, particularly a mammal, more particularly a bovine, comprising a nucleic acid probe or primer having a nucleotide sequence derived from the glycoprotein gE-gene of BHV-1, and a detection means suitable for a nucleic acid detection assay.
Further, the invention relates to a diagnostic kit for detecting antibodies which are specific for BHV-1, in a sample, in particular a biological sample such as blood or blood serum, saliva, sputum, bodily fluid such as tears, lung lavage fluid, nasal fluid, milk, or tissue, coming from an animal, in particular a mammal, more in particular a bovine, comprising glycoprotein gE of BHV-1, a part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex of the glycoproteins gE and gI, and a detection means suitable for an antibody detection assay. Such a diagnostic kit may further comprise one or more antibodies which are specific for glycoprotein gE of BHV-1 or specific for a complex of the glycoproteins gE and gI of BHV-1.
The invention also relates to a diagnostic kit for detecting protein of BHV-1 in a sample, in particular a biological sample such as blood or blood serum, blood cells, mil, bodily fluids such as tears, lung lavage fluid, nasal fluid, sperm, in particular seminal fluid, saliva, sputum of tissue, in particular nervous tissue, coming from an animal, in particular a mammal, more in particular a bovine, comprising one or more antibodies which are specific for glycoprotein gE of BHV-1 or specific for a complex of the glycoproteins gE and gI of BHV-1, and a detection means suitable for a protein detection assay.
The invention further provides a method for determining BHV-1 infection of an animal, in particular a mammal, more in particular a bovine, comprising examining a sample coming from the animal, in particular a biological sample such as blood or blood serum, blood cells, sperm, in particular seminal fluid, saliva, sputum, bodily fluid such as tears, lung lavage fluid, nasal fluid, milk, or tissue, in particular nervous tissue, for the presence of nucleic acid comprising the glycoprotein gE-gene of BHV-1, or the presence of the glycoprotein gE of BHV-1 or a complex of the glycoproteins gE and gI of BHV-1, or the presence of antibodies which are specific for the glycoprotein gE of BHV-1 or specific for a complex of the glycoproteins gE and gI of BHV-1. The sample to be examined can come from an animal which has not been previously vaccinated with a vaccine composition according to the invention or from an animal which has previously been vaccinated with a vaccine preparation according to the invention.
The invention relates to a set of BHV-1 vaccines, both live and inactivated, which have in common that they lack the glycoprotein gE gene in whole or in part. This set comprises both a natural gE deletion mutant and constructed gE deletion mutants which may or may not also comprise a deletion of the thymidine kinase gene and/or the glycoprotein gI gene, and constructed gE deletion mutants which are used as vectors for heterologous genes. The invention further relates to nucleotide sequences encoding the BHV-1 glycoprotein gE-gene, oligonucleotides derived from these sequences, the glycoprotein gE itself, peptides which are derived therefrom and (monoclonal or polyclonal) antibodies which are directed against the gE glycoprotein and peptides derived therefrom. The invention also relates to complexes of the glycoproteins gE and gI of BHV-1, and to antibodies directed against such complexes.
These materials according to the invention can be used for:
1) the vaccination of cattle against diseases caused by BHV-1, such that a distinction can be made between BHV-1 infected animals and vaccinated animals; the conventional and the constructed vaccine can be used side by side;
2) the vaccination of cattle against both BHV-1 diseases and diseases caused by other pathogens of which coding sequences for protective antigens can be incorporated into the BHV-1 deletion mutants;
3) testing blood, serum, milk or other bodily fluids from cattle to determine serologically or by means of nucleic acid detection techniques (e.g. PCR) whether they have been infected by a wild-type BHV-1 or have been vaccinated with a gE deletion mutant.
Synthesis of oligopeptides, polypeptides and glycoproteins derived from the coding sequence of the glycoprotein gE-gene and the glycoprotein gI-gene of BHV-1
The results of the DNA sequence analysis, described in the examples, of the glycoprotein gE-gene (FIG. 3A) and the isolated DNA fragments which code for this gene, make it possible, using standard molecular-biological procedures, both to synthesize peptides of the gE protein (oligo or polypeptides) and to express the gE protein in its entirety or in large parts via the prokaryotic route (in bacteria) or via the eukaryotic route (for instance in murine cells). Via these routes, gE-specific antigen can be obtained which can for instance serve for generating gE-specific monoclonal antibodies (Mabs). Furthermore, gE-specific antigen (and gE-specific Mabs) can be used in serological tests to enable a distinction to be made between animals vaccinated with a BHV-1 gE deletion vaccine and animals infected with wild-type BHV-1 virus.
The results of the partial DNA sequence analysis of the glycoprotein gI genexe2x80x94described in the examplesxe2x80x94and the isolated DNA fragments that code for this gene, together with the eukaryotic cells expressing glycoprotein gE, allow the expression of the gI/gE complex in eukaryotic cells (See FIGS. 13 and 14). This glycoprotein complex can be used to produce gI/gE specific monclonal antibodies. The gI/gE complex can also be used as antigen in serological tests to differentiate between cattle vaccinated with a single gE BHV-1 deletion mutant or with a double gI/gE BHV-1 deletion mutant and cattle infected with wild type BHV-1 virus.
gE specific peptides
On the basis of a known protein coding sequence, by means of an automatic synthesizer, polypeptides of no less than about 40-50 amino acids can be made. Now that the protein coding sequence of the gE glycoprotein of BHV-1 strain Lam has been unraveled (FIG. 3A), polypeptides of this BHV-1 gE glycoprotein can be synthesized. With such polypeptides, according to standard methods, experimental animals such as mice or rabbits can be immunized to generate gE-specific antibodies. Further, using these gE-specific peptides, the locations where anti-gE antibodies react with the gE protein (the epitopes) can be further specified, for instance with the PEPSCAN method (Geysen et al., 1984 Proc. Natl. Acad. Sci. USA 81, 3998-4002). gE specific oligopeptides can also be used in serological tests which demonstrate anti-gE antibodies.
Prokaryotic expression of gE
For the synthesis of the gE protein in bacteria (i.e. the prokaryotic expression of gE), DNA fragments which code for the glycoprotein gE or for parts thereof must be cloned into prokaryotic expression vectors. Prokaryotic expression vectors are circular DNA molecules which can maintain themselves in a bacterium as a separately replicating molecule (plasmid). These expression vectors contain one or more marker genes which code for an antibiotic resistance and thus enable the selection for bacteria with the expression vector. Further, expression vectors comprise a (often controllable) promoter region behind which DNA fragments can be ligated which are then expressed under the influence of the promoter. In many current prokaryotic expression vectors, the desired protein is expressed while fused to a so-called carrier protein. To that end, in the vector there is located behind the promoter the coding sequence for the carrier protein, directly adjacent to which the desired DNA fragment can be ligated. Fusion proteins are often more stable and easier to recognize and/or to isolate. The steady-state level which a particular fusion protein can attain in a certain bacterial strain differs from fusion to fusion and from strain to strain. It is customary to try different combinations.
Eukaryotic expression of the glycoprotein gE-gene
Although prokaryotic expression of proteins offers some advantages, the proteins lack the modifications, such as glycosylation and the like, which occur in eukaryotic cells. As a result, eukaryotically expressed protein is often a more suitable antigen. For the heterologous expression of proteins in eukaryotic cells, such as murine cells, use is made of eukaryotic expression vectors. These vectors are plasmids which can not only be multiplied in E. coli cells but also subsist stably in eukaryotic cells. In addition to a prokaryotic selection marker, they also comprise a eukaryotic selection marker. Analogously to the prokaryotic expression vectors, eukaryotic expression vectors contain a promoter region behind which desired genes can be ligated. However, the promoter sequences in eukaryotic vectors are specific for eukaryotic cells. Moreover, in eukaryotic vectors fusion to carrier proteins is utilized only rarely. These vectors are introduced into the eukaryotic cells by means of a standard transfection method (F. L. Graham and A. J. van der Eb, 1973, Virology 52, 456-467). In addition to the eukaryotic plasmid vectors, there are also viral vectors, where the heterologous gene is introduced into the genome of a virus (e.g. retroviruses, herpesviruses and vaccinia virus). Eukaryotic cells can then be infected with recombinant viruses.
In general, it cannot be predicted what vector and cell type are most suitable for a particular gene product. Mostly, several combinations are tried.
Eukaryotic expression of both the glycoprotein gE and the glycoprotein gI
The final structure that a protein obtains, is depending on its primary amino acid sequence, its folding, its posttranslational modifications etc. An important factor that contributes to structure of a protein is its interaction with one or more other proteins. We have found that also BHV-1 glycoprotein gE forms a complex with at least one other glycoprotein: BHV-1 glycoprotein gI. The first indication for such a complex came from our results with candidate anti-gE Mabs 1, 51, 67 75 and 78 (See table 2). These Mabs did not react with Difivac-1, nor with Lam gExe2x88x92 but also failed to recognize glycoprotein gE-expressing 3T3 cells. However, these Mabs did react with gE-epressing 3T3 cells after infection with Difivac-1, showing that complementing factors are needed to give glycoprotein gE the proper antigenic conformation for these Mabs. In some of our radio-immunoprecipitation experiments with Mab 81 we found coprecipitation of a protein with an apparent molecular weight of 63 kD. In view of the fact that the herpes simplex virus glycoprotein gE forms a complex with a protein with a comparable molecular weight (HSV1 glycoprotein gI), we inferred that BHV-1 glycoprotein gE forms a complex with the BHV-1 homolog of glycoprotein gI. To study this BHV-1 gE/gI complex and to produce gE antigen with the proper antigenic structure we expressed both glycoproteins in one eukaryotic cell. For this we applied the same procedure as described for the eukaryotic expression of glycoprotein gE alone. The only additional prerequisite is the use of expression vectors with different eukaryotic selectable markers.
Serological tests
Serological methods for making a distinction between cattle vaccination with Difivac-1 and cattle infected with wild-type BHV-1 on the basis of antibodies against gE are preferably based on the use of monclonal antibodies directed against gE. These can be used in the following manners:
a) According to the principle described by Van Oirschot et al. (Journal of Virological Methods 22, 191-206, 1988). In this ELISA for the detection of gI antibodies against the virus of Aujeszky""s disease, antibodies are demonstrated by their blocking effect on the reaction of two Mabs having two different epitopes on gI. The test is carried out as follows. Microtiter plates are coated with Mab 1, overnight at 37xc2x0 C., after which they are stored, e.g. at 4xc2x0 C. or xe2x88x9220xc2x0 C. The serum to be examined is preincubated with antigen in separate uncoated microtiter plates, e.g. for 2 h at 37xc2x0 C. The Mab 1-coated plates are washed, e.g. 5 times, after which Mab 2 coupled to horseradish peroxidase (HRPO) is added to these plates. Then the preincubated serum-antigen mixtures are transferred to the plates in which the two Mabs are located, followed by incubation, e.g. for 1 h at 37xc2x0 C. The plates are washed and substrate is added to each well. After e.g. 2 h at room temperature, the plates are spectrophotometrically read. Four negative control sera and four serial dilutions of a positive serum are included on each plate. The serum which has an optical density (OD) value of less than 50% of the average OD value of the 4 negative control sera which have been examined on the same plate, is considered positive.
b) According to the Indirect Double Antibody Sandwich (IDAS) principle. Here, microtiter plates are coated with an Mab or a polyclonal serum directed against the gE protein. Incubation with a gE antigen preparation results in gE binding to the coating. Antibodies specifically directed against gE in the bovine serum to be examined subsequently bind to the gE. These bound antibodies are recognized by an anti-bovine immunoglobulin conjugate. The antibodies in this conjugate are covalently bound to peroxidase enzyme. Finally, the bound conjugate is visualized by addition of a chromogenic substrate. The specificity of the reaction is checked by carrying out the same procedure with a gE-negative control preparation instead of a gE-antigen preparation. On each microtiter plate, positive and negative control sera are included. The test is valid if the positive serum scores positive in a certain dilution. A serum is positive if it scores an OD which is 0.2 higher than the standard negative control serum.
c) According to the IDAS principle as described under 2, but after incubation of the serum to be examined an anti-gE Mab/HRPO is used instead of the anti-bovine immunoglobulin conjugate. An anti-gE peptide serum or an anti-gE polyclonal serum may be used instead of the anti-gE Mab. The plates are washed and to each well a chromogenic substrate is added. After e.g. 2 h at room temperature, the plates are spectrophotometrically read. Four negative control sera and four serial dilutions of a positive serum are included on each plate. The serum which has an OD value of less than 50% of the average OD value of the 4 negative control sera which have been examined on the same plate, is considered positive.
d) According to the principle of a blocking ELISA, whereby virus antigen which may or may not be purified is coated to the microtiter plate overnight. In these plates, the serum to be examined is incubated for, e.g. one hour or longer at 37xc2x0 C. After a washing procedure, an anti-gE Mab is added to the plates, followed by incubation for e.g. 1 h at 37xc2x0 C. An anti-gE peptide serum or an anti-gE polyclonal serum may be used instead of the anti-gE Mab. The plates are washed and to each well a chromogenic substrate is added. After e.g. 2 h at room temperature, the plates are read spectrophotometrically. Four negative control sera and four serial dilutions of a positive serum are included on each plate. The serum which has an OD value of less than 50% of the average OD value of the 4 negative control sera which have been examined on the same plate, is considered positive.
In all the above arrangements, conventionally grown virus antigen which contains gE can be used, but so can gE-antigen which is expressed via prokaryotes or eukaryotes. Alternatively, oligopeptides based on the BHV-1 gE sequence could be used in the above diagnostic tests instead of conventional antigen. In addition, such oligopeptides could be used for the development of a so-called xe2x80x9ccow-sidexe2x80x9d test according to the principle described in an article by Kemp et al., Science 241, 1352-1354, 1988. Such a test would then be based on a binding of the antigenic sequence of the oligopeptide by antibodies directed against gE, present in infected animals. For such a test, the oligopeptide would have to be coupled to an Mab directed against bovine erythrocytes.
Nucleic acid analysis using the polymerase chain reaction
Oligonucleotides (probes and primers) can for instance be used in the polymerase chain reaction to make a distinction between vaccinated and infected animals. The polymerase chain reaction (PCR) is a technique whereby nucleic acids of a pathogen can be multiplied billions of times in a short time (De polymerase kettingreactie, P. F. Hilderink, J. A. Wagenaar, J. W. B. van der Giessen and B. A. M. van der Zeijst, 1990, Tijdschrift voor Diergeneeskunde deel 115, 1111-1117). The gE oligonucleotides can be chosen such that in a gE positive genome a different product is formed than in a gE negative genome. The advantage of this is that also an animal which has been vaccinated with a gE deletion vaccine gives a positive signal in a PCR test. However, this approach depends on the presence of nucleic acids of the virus in a sample, for instance blood, coming from the animal to be tested.
After an acute BHV-1 infection, there is a great chance that BHV-1 specific nucelic acids can be demonstrated in the blood, but it has not been determined yet whether BHV-1 nucleic acids can also be demonstrated in the blood during latency.
The use of BHV-1 as a vector
For expressing heterologous genes in the BHV-1 genome, it is necessary to have disposal of exact information on the area where the heterologous gene is to be inserted. There should not be any disturbance of essential sequences, and regulatory sequences must be available for the expression of the heterologous gene. In principle, the glycoprotein gE-gene is a suitable place to express heterologous genes. The gE-gene is not essential, hence there is no objection to replacing the gE gene by the heterologous gene. As a consequence, the heterologous gene can be so positioned that it will be under the influence of the regulatory sequences of the gE gene. However, it is not necessary to use the regulatory sequences of the gE-gene. The expression of heterologous genes may be controlled alternatively by other, e.g. stronger regulatory sequences of different genes. It is also possible to ligate the heterologous gene to the (export) signal peptide of the gE gene, so that the secretion of the heterologous gene product can be influenced. It is clear that detailed knowledge of the gE gene and the gE protein affords the possibility of using BHV-1 as a vector in a very measured manner. The vectors developed can moreover be serologically distinguished from wild-type. The construction of BHV-1 mutants which express heterologous genes can be carried out in the same manner as the construction of gE deletion mutants shown in the examples. However, the deletion fragments should then be replaced with a fragment on which a heterologous gene is located at the location of the deletion.